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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
dc.contributor.author | Chang-Ting Tsai | en |
dc.contributor.author | 蔡昌廷 | zh_TW |
dc.date.accessioned | 2021-06-15T04:25:13Z | - |
dc.date.available | 2014-09-02 | |
dc.date.copyright | 2009-09-02 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-21 | |
dc.identifier.citation | 1. Terpe K: Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 2006, 72(2):211-222.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45526 | - |
dc.description.abstract | 本研究利用醱酵生產的調控方式,提高嗜甲醇酵母菌Pichia pastoris及Pichia methanolica生產重組木聚醣酶之產量,並利用Saccharomyces cerevisiae的IRES建構嗜甲醇酵母菌雙順反子表現質體,於Hansenula polymorpha中成功地表現位於IRES下游的綠色螢光蛋白質。在醱酵生產的調控上,P. pastoris利用高密度細胞培養且在誘導前置換培養基,並維持誘導時甲醇濃度於0.5%可以得到最高的木聚醣酶活性,在甲醇誘導後10天木聚醣酶活性為5400 U/ml。而在P. methanolica中利用全合成培養基,並以1.8 ml/h/l固定流速連續添加甲醇的方式進行誘導,可以得到最高的木聚醣酶活性,在甲醇誘導後10天木聚醣酶活性為6200 U/ml。在P. methanolica誘導策略的研究上,固定流速連續添加甲醇的模式較依溶氧曲線逐段提高甲醇添加量及利用MC168維持甲醇濃度恆定的模式,在誘導時所需的的甲醇量最少而木聚醣酶活性最高,且菌體生長的數目最少。分析P. pastoris及P. methanolica表現的重組木聚醣酶可知兩者皆有不錯的熱穩定性及pH穩定性。利用SDS-PAGE及西方雜合法分析二者生產的重組木聚醣酶,可知其胞外上清液內的主要成分皆為木聚醣酶,以醣蛋白染色法可以確認二者所生產的重組木聚糖酶皆有糖基化修飾,然而此種糖基化修飾可能是較為複雜的O端糖基化修飾。總而言之,P. methanolica有較簡便的醱酵及誘導程序,並可以得到較高木聚醣酶活性,然而其胞外雜蛋白較多,而P. pastoris的生產的木聚醣酶活性稍低但胞外雜蛋白較少有利於後續的分析純化及應用。此外,利用P. methanolica之pulse-chase實驗可以觀察到在甲醇添加、鹼的添加及木聚糖酶活性上有一密切的關係。此外,為了能夠提高嗜甲醇酵母菌的應用性,本研究建構一個可以用於嗜甲醇酵母菌,以AOX1為啟動子並以木聚糖酶及綠色螢光蛋白質 (EGFP) 為報導基因,並在兩個基因之間加入一個可以替換不同來源IRES (internal ribosome entry site) 之MCS (multiple cloning site) 的雙順反子表現質體,測試來自Saccharomyces cerevisiae的p150、YAP1、TFIID、HAP4 之5’端未轉譯區的IRES序列。在H. polymorpha內發現來自YAP1、TFIID、HAP4之IRES皆具有活性,並成功地使IRES下游的EGFP表現。利用西方雜合法及螢光顯微鏡可明顯地觀察到EGFP訊號,然而來自p150之IRES在H. polymorpha內僅能使EGFP少量地表現,而在P. pastoris內並無法偵測得到EGFP的表現。 | zh_TW |
dc.description.abstract | In this study, the xylanase gene from the rumen fungus Neocallimastix frontalis was expressed in Pichia pastoris and Pichia methanolica. In the study of methanol induction strategy of P. methanolica, the highest xylanase activity, fewest amounts of methanol feeding, and least cell growth was made by using continuous methanol feeding strategy. Using high cell density culture with medium replacement before induction and the maintenance of the methanol induction level at 0.5%, P. pastoris was able to produce about 5400 U/ml of xylanase after 10 days of induction. For P. methanolica, about 6200 U/ml of xylanase was reached after 10 days of induction using synthetic medium as first culture medium and then direct induction by continuous methanol feed at 1.8 ml/l/h. In general, the advantages of using P. methanolica to produce the xylanase were higher protein production, the lack of medium replacement, and an ease of scale up. However, due to the culture supernatant with fewer secreted non-target proteins, xylanase purification in P. pastoris would be easier than in the P. methanolica system. The recombinant xylanases from P. pastoris and P. methanolica were glycoproteins, which were in complex O-link glycosylation and had good thermostability and pH stability. In addition, methanol pulses-chase experiment suggested that these was a corelation among base feeding, methanol consumption and xylanase activity. In addition, in order to improve the application of methyltrophic yeast by dicistronic expression plasmid, a methyltrophic yeast dicistronic expression vector was constructed based on the 5’UTR internal ribosome entry site (IRES) of the Saccharomyces cerevisiae p150, YAP1, TFIID, HAP4. Expression vector containing the Neocallimastix frontallis xylanase gene and green fluorescent protein gene flanking the yeast 5’UTR IRES can produce recombinant xylanase and fluorescence in methyltrophic yeast Hansenula polymorpha under the control of alcohol oxidase1 (AOX1) promoter. After methanol induction for three days, the green fluorescence in H. polymorpha was observed under fluorescence microscope. From Western blot analysis, the expression of green fluorescent protein was confirmed. Unfortunately, Pichia pastoris containing this dicistronic plasmid with p150 5’UTR IRES can not produce green fluorescent protein. These results suggest that the S. cerevisiae p150, YAP1, TFIID, HAP4 IRES can be used in the development of dicistronic expression vectors for production of heterologous multiprotein complexes, or can be used for selection markers to facilitate the applications of H. polymorpha. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:25:13Z (GMT). No. of bitstreams: 1 ntu-98-D92b47403-1.pdf: 5210174 bytes, checksum: 4b89542daa798d581a090d0dd9c699e2 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目 錄
摘要................................................................................................................................................II ABSTRACT..................................................................................................................................III 圖目錄 ……………………………………………………………………………………..…VIII 表目錄 …………………………………………………………………………………………..X 1.前言……………………………………………………...……….….…………………………1 1.1異源表現系統……………………………………………………………………………...1 1.2酵母菌表現系統………………………………………………………………………...…2 1.3嗜甲醇酵母菌表現系統...……………………………….…..………………………….....3 1.3.1 P. pastoris………….…………………………………………………………………6 1.3.2 H. polymorpha………...…………………………………………………………….10 1.3.3 P. methanolica……………………………...……………………………………….13 1.4重組蛋白質的大規模表現……………………......……………………….………..……16 1.5真核生物的轉譯起始與Internal ribosome entry site (IRES)……………………............17 1.6酵母菌的IRES………………………..…………………………………………………..28 1.7瘤胃真菌Neocallimastix frontallis的木聚醣酶……………………………………..…..30 1.8綠色螢光蛋白質……………………………………………….………………….………35 2.研究動機、實驗構想及具體目標…………………………………………………………….37 2.1研究動機…………………………………………………………………………...………37 2.2實驗構想…………………………………………………………………………...………37 2.2.1利用不同的培養及誘導策略提高重組蛋白質產量產………………………..……37 2.2.1.1 P. pastoris細胞高密度培養……………………………………...….……...37 2.2.1.2 P. methanolica不同誘導條件之探討……………………………..………..38 2.2.1.3具體目標………………………………………………………...…………..39 2.2.2 IRES於嗜甲醇酵母菌之應用.…………………………………………..….………41 2.2.2.1建構嗜甲醇酵母菌表現系統的IRES雙順反子表現質體….……..………41 2.2.2.2利用IRES進行多順反子表現………………………….…………………..41 2.2.2.3具體目標…………………………………………………...………………..43 3.材料與方法………………………………………………………………..………………….45 3.1菌株與質體..…………………………………………….…………………………….…..45 3.2 儀器..…………………………………………….…………………………….………....48 3.3聚合酶鏈索反應(PCR)………..…..…………………………………..…………………..49 3.3.1試劑、引子及PCR條件……….……………………………………….…………..49 3.3.2 Overlap PCR………………………………………………………..……………….49 3.3.3 PCR產物分析……………..………………………………….……………….……49 3.4質體建構…………………………………………………………………………….….….50 3.4.1質體DNA抽取、overlap PCR及限制酶截切……………………………….……....50 3.4.2 S. cerevisiae 5’UTR IRES選殖及建構嗜甲醇酵母菌雙順反子表現質體….….….50 3.5質體轉形………………………………………………………………………………...…53 3.5.1 P. pastoris電穿孔勝任細胞的製備…………………………...…………………….53 3.5.1.1電穿孔轉形法…………………………………………………...……………53 3.5.2 H. polymorpha電穿孔勝任細胞的製備…………………………………………….53 3.5.2.1電穿孔轉形法…………………….…………………………………..………54 3.6轉形株篩選…………………………………….…….…………………………………….54 3.6.1轉形株利用小量培養篩選………………………………………………..…………54 3.7嗜甲醇酵母菌之醱酵槽培養及生產…………………………………………...………..54 3.7.1批次培養……………………………………………………....……………………54 3.7.2批次饋料培養……………………………………………...……………………….55 3.7.3甲醇誘導……………………………………………………..……………………..55 3.8生長曲線之測定與計算…………………………………………………………….……55 3.9木聚醣酶活性測定………….……………………………………………………………55 3.9.1活性測定方法……………………..……………………………..…………...…….56 3.10蛋白質電泳分析………………...…………………………...……………………...…..57 3.11西方墨點法分析……...……………………………….……..….……….…………..….58 3.12 醣蛋白質染色法……………………………………………………………………….59 4.結果.………………………………………………...………………………………………...60 4.1 P. pastoris最適誘導條件之探討………………………………...………………...…..60 4.1.1利用細胞高密度培養提高P. pastoris重組木聚醣酶活性…..……………….….60 4.2 P. methanolica最適誘導條件之探討…….………………………….…………………..62 4.2.1利用不同甲醇誘導策略提高P. methanolica重組木聚醣酶表現量.........………..62 4.2.2 P. methanolica利用甲醇連續誘導生產重組木聚醣酶....……………..……….…66 4.3醱酵參數之探討…………………………………….……………………………………68 4.3.1 P. pastori之醱酵參數…………………………………………...…….………….68 4.3.2 P. methanolica之醱酵參數………………………………………………………...68 4.3.3 P. methanolica之pulse-chase實驗……………………….……..……………….....68 4.4 SDS-PAGE及西方墨點法分析…..…………………………………………………...…71 4.5醣蛋白質染色及去醣基酵素截切……………………..……….………………………..74 4.6重組木聚醣酶熱穩定性及pH穩定性分析……………….……….……………………77 4.7嗜甲醇酵母菌的雙順反子表現…………………………….……………………………80 4.7.1嗜甲醇酵母菌雙順反子表現質體的建構……….………………………….……..80 4.7.1.1報導基因選殖及overlap PCR……………………….………..………..….80 4.7.1.2報導基因片段與表現質體的限制酶截切及黏合……………………..….81 4.7.1.3 S. cerevisiae的IRES之選殖、雙順反子表現質體建構及定序確認……..85 4.7.2 S. cerevisiae p150 IRES於P. pastoris的作用………………………….………….92 4.7.3 S. cerevisiae p150 IRES於H. polymorpha的作用………………….…….……….94 4.7.4 S. cerevisiae YAP1、TFIID、HAP4 IRES於H. polymorpha的作用……..……….96 5.討論 ..……………………………………………………………………………………….101 5.1嗜甲醇酵母菌表現系統醱酵策略之探討……………………………………………...101 5.2嗜甲醇酵母菌表現系統P. pastoris與P. methanolica之比較........................................103 5.3重組木聚醣酶之應用………………………………………….......................................107 5.4含有S. cerevisiae IRES的雙順反子表現質體於嗜甲醇酵母菌P. pastoris與 H. polymorpha中表現之探討…………………………………………………………..111 5.5 雙順反子表現質體……………………………………………………………………..112 5.6 未知IRES的篩選………………………………………………………………………113 6.總結…………………………………………………………………………………….…….115 7.未來工作與展望 ………………………………………………………………………........117 8.參考文獻……………………………………………………………………………………..119 9.附錄………………………………………………………………………………………….136 本研究已發表文獻…………………………………………………………………………136 圖目錄 圖一、嗜甲醇酵母菌之甲醇利用途徑…………………………………………….…………...5 圖二、原核生物的轉譯起始…………………………………….……………………..……….20圖三、真核生物的轉譯起始…………………………………….……………………..……….21 圖四、cap-dependent轉譯起始與4群病毒IRES轉譯起始所需的反式作用因子…………22 圖五、比較真核生物的cap-dependent轉譯起始與IRES轉譯起始…………………………23 圖六、細胞壁組成及各種纖維分解酵素作用示意圖…………………………………………33 圖七、木聚醣結構及各木聚醣分解酵素作用位置示意圖…………………………………...34 圖八、綠色螢光蛋白質之結構、發色團及吸收和放射光譜……………………………..……36 圖九、利用醱酵槽進行嗜甲醇酵母菌大規模之重組蛋白質生產之研究架構圖…………....40 圖十、利用IRES提高嗜甲醇酵母菌轉譯效率及雙順反子表現之研究架構圖….………….44 圖十一、嗜甲醇酵母菌雙順反子質體建構示意圖……………………………………..…….51 圖十二、P. pastoris之細胞高密度培養………...…………….………………………...……...61 圖十三、P. methanolica以固定速率連續添加甲醇誘導 (A誘導模式)………………..……..63 圖十四、P. methanolica以連續添加逐段提高甲醇添加速率誘導 (B誘導模式)....................64 圖十五、P. methanolica以甲醇監控儀維持甲醇濃度恆定誘導 (C誘導模式)........................65 圖十六、P. methanolica以固定速率連續添加甲醇誘導…………….......................................67 圖十七、P. methanolica的pulse-chase實驗.………..................................................................70 圖十八、利用13 % SDS-PAGE以CBR染色分析P. pastoris YS27及P. methanolica M6-16不同誘導時間之樣品…………..…..……………………….………………………….………72 圖十九、以西方墨點法分析P. pastoris YS27及P. methanolica M6-16 之胞外上清液……………..……………………………………................................73 圖二十、利用醣蛋白質染色法分析 P. pastoris YS27及 P. methanolica M6-16 之胞外上清 液……………………………………………………………………………………..75 圖二十一、利用去醣基酵素處理P. pastoris YS27及P. methanolica M6-16之重組木聚醣 酶………………………………………………………………………………..…76 圖二十二、P. pastoris YS27及P. methanolica M6-16重組木聚醣酶熱穩定性測試…………78 圖二十三、P. pastoris YS27及P. methanolica M6-16重組木聚醣酶pH穩定性測試….…..79 圖二十四、報導基因選殖及overlap PCR……………………………………………..……….82 圖二十五、報導基因片段與嗜甲醇酵母菌表現質體的限制酶截切及黏合…………....……83 圖二十六、嗜甲醇酵母菌雙順反子表現質體之建構………………………………….……..84 圖二十七、 S. cerevisiae之IRES選殖及雙順反子表現質體建構…….……………………86 圖二十八、嗜甲醇酵母菌雙順反子表現質體建構示意圖……………………………………87 圖二十九、p150序列比對……………………………………………………………….…….88 圖三十、YAP1序列比對………………………………………….……..……………………..89 圖三十一、TFIID序列比對……………………………………………………………….…….90 圖三十二、HAP4序列比對………………………………………………………….………….91 圖三十三、P. pastoris之雙順反子表現……………………………………………………….93 圖三十四、 H. polymorpha之雙順反子表現…………………………………………………95 圖三十五、H. polymorpha轉形株木聚醣酶活性測定………………………………….…….97 圖三十六、利用螢光顯微鏡觀察綠色螢光蛋白質之雙順反子表現…………………………98 圖三十七、利用螢光顯微鏡觀察綠色螢光蛋白質之雙順反子表現…………………………99 圖三十八、利用西方墨點法分析雙順反子表現…………………………………………..…100 圖三十九、嗜甲醇酵母菌過氧化物酶體內之甲醇代謝路徑 ………………………………108 表目錄 表一、利用P. Pastoris表現之重組蛋白質……........……………………………………………9 表二、利用H. polymorpha表現之重組蛋白質……………………………….………………12 表三、利用P. methanolica表現之重組蛋白質...........................................................................15 表四、已知含有IRES之病毒………………………………………………………….………..24 表五、病毒的IRES及其所需的ITAF……………………………………………….………..25 表六、細胞內IRES的功能及其所需的ITAF……………………………………….………..26 表七、本研究中所使用菌株....…………………………………………………………………46 表八、本研究中所使用的質體…………………………………………………………………47 表九、本研究中所使用的引子與PCR反應條件…….………….………………………..…..52 表十、酵母菌表現系統之比較 ....……………………………………………………………109 表十一、嗜甲醇酵母菌甲醇代謝相關基因在不同碳源下的反應………………………….110 | |
dc.language.iso | zh-TW | |
dc.title | 利用嗜甲醇酵母菌進行重組蛋白質表現之醱酵生產及分子調控之研究 | zh_TW |
dc.title | The study of recombinant protein expression by fermentation and molecular control in methyltrophic yeasts | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 楊盛行,許瑞祥,陳浩仁,李昆達 | |
dc.subject.keyword | 嗜甲醇酵母菌表現系統,重組蛋白質表現,醱酵生產,分子調控,轉譯調控, | zh_TW |
dc.subject.keyword | methyltrophic yeast expression systems,recombinant protein expression,fermentation,molecular control,IRES,translational control, | en |
dc.relation.page | 144 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-24 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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